Compounds useful as antiproliferative agents and GARFT inhibitors

ABSTRACT

Compounds of the Formula I, which are in equilibrium with their 4-hydroxy tautomers and are in the form of diastereomeric mixtures, and their pharmaceutically acceptable salts are potent GARFT inhibitors: ##STR1## wherein A, Z, X, and R 1  and R 2  are defined in the specification. These compounds and their salts are useful as antiproliferative agents. The invention also pertains to pharmaceutical compositions and methods employing such compounds as GARFT inhibitors or antiproliferative agents. The invention also relates to compounds useful as intermediates for preparing such compounds, and to their synthesis.

BACKGROUND OF THE INVENTION

The present invention relates to compounds of the Formula I definedbelow, which inhibit the enzyme glycinamide ribonucleotide formyltransferase (GARFT). The invention also relates to pharmaceuticalcompositions containing the compounds of the Formula I, to their use toinhibit GARFT and to their use to inhibit the growth and proliferationof the cells of higher organisms or microorganisms such as bacteria,yeast and fungi. The invention also relates to the preparation of thesecompounds, and to intermediates used in their preparation.

GARFT is a folate dependent enzyme in the de novo purine biosynthesispathway. This pathway is critical to cell division and proliferation.Shutting down this pathway is known to have an antiproliferative effect,in particular, an antitumor effect. Thus, a number of folate analogshave been synthesized and studied for their ability to inhibit GARFT. Aprototypical specific tight-binding inhibitor of GARFT,5,10-dideazatetrahydrofolic acid (DDATHF), has been reported to showantitumor activity. See F. M. Muggia, "Folate antimetabolites inhibitorto de novo purine synthesis," New Drugs, Concepts and Results in CancerChemotherapy, Kluwer Academic Publishers, Boston (1992), 65-87.

The large class of antiproliferative agents includes antimetabolitecompounds. A particular subclass of antimetabolites known as antifolatesor antifoles are antagonists of the vitamin folic acid. Typically,antifolates closely resemble the structure of folic acid and incorporatethe characteristic P-benzoyl glutamate moiety of folic acid. Theglutamate moiety of folic acid takes on a double negative charge atphysiological pH, and therefore this compound and its analogs have anactive energy driven transport system to cross the cell membrane andexert a metabolic effect. Research by a number of investigators has showthat folic acid in both its reduced and oxidized forms and its analogsare actively transported into cells by at least two distinct transportmechanisms. These transport proteins are referred to as the reducedfolate transport protein, which has a preference for reduced folates butwill transport a number of folic acid derivatives. Methotrexate (MTX) istransported via the reduced folate transport system. The other folatetransport protein is referred to as the membrane folate binding proteinor mFBP, which has a preference for folic acid. See A. C. Antony, "TheBiological Chemistry of Folate Receptors," Blood, The Journal of theAmerican Society of Hematology, vol. 79 (1992), 2807-2820.

The anticancer glutamate-containing antifolates used clinically to date,including MTX, enter cells via the reduced folate transport system withone notable exception. 5,10-Dideazatetrahydrofolic acid (DDATHF) is anantitumor GARFT inhibitor currently undergoing clinical study. DDATHFhas been shown to be transported into cells via both the reduced folatetransport system and the mFBP. See G. Pizzorno et al.,"5,10-Dideazatetrahydrofolic Acid (DDATHF) Transport in CCRF-CEM andMA104 Cell Lines," The Journal of Biological Chemistry, vol. 268 (1993),1017-1023.

It has been suggested that undesirable toxicity, particularly infolate-depleted mammals, is related to the fact that DDATHF, a prior artGARFT inhibitor, has a high affinity for the mFBP, which is unregulatedduring times of folate deficiency. It has been further suggested thatfolic acid and other molecules that block the mFBP from transportingother GARFT inhibitors can attenuate the toxicity of such inhibitors.See, e.g., T. Alati et al., "Evaluation of the Mechanism(s) ofInhibition of the Toxicity, But Not the Antitumor Activity of Lometrexol(DDATHF) by Folic Acid," Proceedings of the American Association forCancer Research, vol. 33 (1992), Abstract 2432, 407; L. L. Habeck etal., "A Novel Class of Monoglutamated Antifolates Exhibits Tight-bindingInhibition of Human Glycinamide Ribonucleotide Formyltransferase andPotent Activity against Solid Tumors," Cancer Research, vol. 54 (1994),1021-1026; and U.S. Pat. No. 5,217,974 to Grindey et al.

SUMMARY OF THE INVENTION

Thus, an object of this invention is to produce compounds that arepotent GARFT inhibitors having reduced toxicity. This object has beenachieved through the antiproliferative agents of the Formula I belowthat are potent GARFT inhibitors but do not have tight binding to themFBP. These compounds preferably have binding constants to the mFBP ofat least a factor of 1000 less than DDATHF, yet still retain thefavorable properties of GARFT inhibition and reduced folate transportfor antitumor activity.

As indicated above, compounds of the invention possess antiproliferativeactivity, a property which can express itself in the form of antitumoractivity. A compound of the invention can be active per se, or as aprecursor converted in vivo to an active compound. Preferred compoundsof the invention are especially active in inhibiting the enzyme GARFT.Particularly preferred compounds are active in inhibiting the growth ofthe L1210 cell line, a mouse leukemia cell line that can be grown intissue culture. Compounds of the invention can also be active ininhibiting the growth of bacteria such as Escherichia coli gram-negativebacteria which can be grown in culture.

The compounds according to the invention, as well as thepharmaceutically acceptable salts thereof, may be incorporated intoconvenient dosage forms, such as capsules, tablets and injectablepreparations. Solid or liquid pharmaceutically acceptable carriers,diluents or excipients may also be employed.

Solid carriers include starch, lactose, calcium sulfate dihydrate, terraalba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearateand stearic acid. Liquid carriers include syrup, peanut oil, olive oil,saline solution and water.

The carrier or diluent may include any prolonged-release material, suchas glyceryl monostearate or glyceryl distearate, alone or with wax. Whena liquid carrier is used, the preparation may be in the form of a syrup,elixir, emulsion, soft gelatin capsule, sterile injectable liquid (e.g.solution) or a nonaqueous or aqueous liquid suspension.

The pharmaceutical preparations are prepared following conventionaltechniques of the pharmaceutical chemist involving steps such as mixing,granulation and compressing when necessary for tablet forms, or mixing,filling and dissolving the ingredients as appropriate to give thedesired products for oral, parenteral, topical, intravaginal,intranasal, intrabronchial, intraocular, intraaural or rectaladministration.

The compositions of the invention may further comprise one or more otherpharmaceutically active compounds. For example, one of the followingantitumor agents may be included in the composition: mitotic inhibitors(e.g., vinblastine); alkylating agents; dihydrofolate reductaseinhibitors or TS inhibitors; antimetabolites (for example,5-fluorouracil, cytosinerabinoside); intercalating antibiotics (forexample, adriamycin, bleomycin); enzymes (for example, asparaginase);topoisomerase inhibitors (for example, etoposide); and biologicalresponse modifiers (for example, interferon). The compounds of theinvention may also be used in combination with one or moreantiproliferative agents or GARFT inhibitors, such as a compounddescribed in commonly assigned International Publication No. WO94/13295, published Jun. 23, 1994, or International Publication No. WO92/05153, published Apr. 2, 1992, the disclosures of which areincorporated by reference herein. The compositions of the invention mayalso comprise one or more antibacterial, antifungal, antiparasitic,antiviral, antipsoriatic or anticoccidial agents. Exemplaryantibacterial agents include: sulfonamides, such as sulfamethoxazole,sulfadiazine, sulfameter and sulfadoxine; dihydrofolic reductaseinhibitors, such as trimethoprim, bromodiaprim and trimetrexate;penicillins; cephalosporins; and the quinolone carboxylic acids andtheir fused isothiazolo analogs.

Another aspect of the invention relates to a therapeutic method ofinhibiting the growth or proliferation of cells of higher organisms ormicroorganisms, which comprises administering to a host an effectiveamount or quantity of a compound according to the present invention. Thecompounds of the invention are particularly useful in the treatment ofmammalian hosts, such as human hosts, and in the treatment of avianhosts. A particularly preferred therapeutic process comprisesadministering to a host an amount of a compound according to the presentinvention effective to inhibit GARFT.

Many of the antiproliferative compounds described herein and theirpharmaceutically acceptable salts thereof can be employed in thetherapeutic process of the invention. The compounds may be administeredin the form of a pharmaceutically acceptable composition comprising adiluent or carrier as described above.

A dose of a composition contains at least an effective quantity of theactive compound and preferably is made up of one or more pharmaceuticaldosage units. An "effective quantity" means a quantity sufficient toinhibit the folate metabolic pathways and derive the beneficial effectstherefrom, e.g., through administration of one or more of thepharmaceutical dosage units.

An exemplary daily dose for a vertebrate host comprises an amount of upto one gram active compound per kilogram of the host, preferablyone-half of a gram, more preferably 100 milligrams, and most preferably,about 50 milligrams or less, per kilogram of the host's body weight. Theselected dose may be administered to a warmblooded animal or mammal, forexample, a human patient in need of treatment mediated by folatemetabolic pathways inhibition, by any suitable method of administratingthe dose including: topically, for example, as an ointment or cream;orally; rectally, for example, as a suppository; parenterally byinjection; or continuously by intravaginal, intranasal, intrabronchial,intraaural or intraocular infusion.

The compounds according to the invention produce any one or more of anantiproliferative effect, an antibacterial effect, an antiparasiticeffect, an antiviral effect, an antipsoriatic effect, an antiprotozoaleffect, an anticoccidial effect, an antiinflammatory effect, animmunosuppressive effect and an antifungal effect. The compounds areespecially useful in producing an antitumor effect in a vertebrate hostharboring a tumor.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

In particular, the invention relates to compounds of the Formula I:##STR2## wherein: A is sulfur, CH₂ or selenium;

Z is a substituted or unsubstituted C₁ -C₃ alkyl group, a substituted orunsubstituted C₂ -C₃ alkenyl group, a substituted or unsubstituted C₂-C₃ alkynyl group, a substituted or unsubstituted amino group, sulfur oroxygen;

X is a substituted or unsubstituted C₁ -C₆ alkyl group; a substituted orunsubstituted C₂ -C₆ alkenyl group; a substituted or unsubstituted C₂-C₆ alkynyl group; --C(O)E, wherein E is hydrogen, a substituted orunsubstituted C₁ -C₃ alkyl group, a substituted or unsubstituted C₂ -C₃alkenyl group, a substituted or unsubstituted C₂ -C₃ alkynyl group, asubstituted or unsubstituted OC₁ -C₃ alkoxy group, or NR₁₀ R₁₁, whereinR₁₀ and R₁₁ are independently selected from hydrogen, substituted andunsubstituted C₁ -C₃ alkyl groups, substituted and unsubstituted C₂ -C₃alkenyl groups, substituted and unsubstituted C₂ -C₃ alkynyl groups;NR₁₀ R₁₁, wherein R₁₀ and R₁₁ are independently defined as set forthabove; hydroxyl; nitro; SR₁₂, wherein R₁₂ is hydrogen, a substituted orunsubstituted C₁ -C₆ alkyl group, a substituted or unsubstituted C₂ -C₆alkenyl group, or a substituted or unsubstituted C₂ -C₆ alkynyl group;cyano; or a substituted or unsubstituted O(C₁ -C₃) group; and

R₁ and R₂ are each independently hydrogen or a moiety that forms(together with the attached CO₂) a readily hydrolyzable ester group.

The invention also relates to pharmaceutically acceptable salts of thecompounds of Formula I.

Although the compounds of the Formula I are shown in the 4-oxo form andare referred to as such throughout this description, the oxo groupexists in tautomeric equilibrium with the corresponding 4-hydroxy group.It will therefore be understood that the compounds of the Formula Iinclude the structurally depicted 4-oxo and the tautomeric 4-hydroxyforms. Thus, the invention also relates to pharmaceutically acceptablesalts of the 4-hydroxy tautomers of the compounds depicted by Formula I.

The compounds of the Formula I are in the form of diastereomericmixtures. It will be understood that unless indicated otherwise, thecompounds having chiral centers are in the form of mixtures ofdiastereomers.

Preferably, A is sulfur or CH₂.

When Z is substituted, the substituents are preferably selected fromC₁₋₆ alkoxyl, C₁₋₆ alkyl and C₂₋₆ alkenyl such as vinyl, C₂₋₆ alkynyl,acyl such as formyl and acetyl, halogen, amino, hydroxyl, nitro,mercapto, monocyclic carbocycle, monocyclic heterocycle, nonfusedpolycyclic carbocycle, nonfused polycyclic heterocycle, hydroxy C₁₋₆alkyl such as hydroxymethyl, and C₁₋₆ alkoxy C₁₋₆ alkyl. Preferably, Zis CH₂, CH₂ CH₂, NH, oxygen, sulfur, CH(CH₂ OH) or NCH₃. Morepreferably, Z is CH₂.

When X is substituted, the substituents are preferably selected from OH,NH₂, O-methyl, O-ethyl, SH, SCH₃ and NH-methyl. Preferably, X is asubstituted or unsubstituted C₁ -C₆ alkyl group. Also, X is preferablyunsubstituted. More preferably, X is methyl or ethyl.

Preferably, R₁ and R₂ each is independently hydrogen, C₁ -C₆ alkyl,hydroxyalkyl, alkylaryl or aralkyl. More preferably, R₁ and R₂ each isindependently hydrogen or C₁ -C₂ alkyl.

In particularly preferred embodiments, A is sulfur or CH₂, Z is CH₂, andX is methyl.

Preferred examples of compounds of the Formula I include:N-(5-[2-(2-amino-4(3H)-oxo-5,6,7,8-tetrahydropyrido[2,3-d]-pyrimidin-6-yl)ethyl]-4-methylthieno-2-yl)-L-glutamicacid;N-(5-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-6][1,4]-thiazin-6-yl)ethyl]-4-methylthieno-2-yl)-L-glutamicacid diethyl ester; andN-(5-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-6][1,4]-thiazin-6-yl)ethyl]-4-methylthieno-2-yl)-L-glutamicacid.

The compounds of the Formula I are useful as GARFT inhibitors. Thecompounds of Formula I in which R₁ and R₂ are each hydrogen areespecially active antitumor or antiproliferative agents. The compoundsof Formula I wherein R₁ and R₂ are each a moiety that forms a readilyhydrolyzable ester group with the attached carboxyl, preferably an ethylgroup, are useful intermediates for forming the free glutamic acid formsof the compounds and can also be hydrolyzed in vivo and thus act asprodrugs.

The pharmaceutically acceptable salts of the invention include, forexample, alkaline metal, alkaline earth metal, other non-toxic metals,and ammonium and substituted ammonium salts of the glutamic acidcompounds of the invention. Exemplary salts include sodium, potassium,lithium, calcium, magnesium, pyridinium and substituted pyridinium saltsof the free acid compounds.

The compounds of the Formula I can be prepared as described below.

To prepare compounds of the Formula I where Z is CH₂, a useful startingmaterial is a compound of the Formula II: ##STR3## wherein: R is ahalogen, preferably bromo; X is as defined above; and B is OH or anamino acid, preferably diethyl glutamate, linked through the aminoportion to form an amide, or a C₁ -C₆ alcohol, preferably a methyl orethyl alcohol, linked through the alcohol portion to form an ester.

The compound of the Formula II is reacted with a compound of the FormulaIII: ##STR4## wherein: Y is CH₂ OH or a protected pyridopyrimidine ofthe Formula IV: ##STR5## The synthesis then can follow one of tworoutes, depending on whether Y is a protected pyridopyrimidine or CH₂OH.

Where Y is a protected pyridopyrimidine or CH₂ OH of the Formula IV, thecoupling reaction of compounds of the Formulae II and III is preferablyconducted in the presence of a transition metal catalyst, preferablypalladium or nickel, in the presence of a base, preferably anon-nucleophilic auxiliary base, in a solvent in which at least one ofthe reactants is at least partially soluble. Preferred solvents for thecoupling reaction of the compounds of Formulae II and III arediethylamine, acetonitrile, dimethylformamide, dimethylacetamide andtriethylamine. The basic medium for the coupling reaction is preferablyprovided via a non-nucleophilic auxiliary base, which is a base capableof neutralizing hydrogen halide acid generated by the coupling reaction.The base is preferably a di- or tri-alkylamine, such as diethylamine,triethylamine or diisopropylethylamine. Where appropriate, a basicsolvent can be used instead of a separate solvent and base.

When Y is the pyridopyridimine the coupling reaction of the compounds ofFormulae II and III produces a compound of the Formula V: ##STR6##wherein X, R₁ and R₂ are as defined above.

The compound of the Formula V is reacted with hydrogen gas, preferablyat 45-1000 psi, in the presence of a suitable transition metal catalyst,preferably platinum, palladium or rhodium metal on a carbon or othersuitable support, in a suitable solvent, preferably acetic acid ortrifluoroacetic acid, to obtain a compound of the Formula VI: ##STR7##wherein X, R₁, and R₂ are defined above.

Finally, the compound of Formula VI is hydrolyzed to form a freeglutamic acid (R₁ and R₂ are each H) of Formula I.

Where Y is CH₂ OH, the reaction of the compounds of Formulae II and IIIproduces a compound of the Formula VII: ##STR8## wherein X and B are asdefined above.

The compound of the Formula VII is reacted with hydrogen gas in thepresence of a suitable metal catalyst, preferably palladium or platinum,to obtain a compound of the Formula VIII: ##STR9## wherein X and B areas defined above.

The compound of the Formula VIII is reacted with an oxidizing agent,preferably tetrapropylammonium perruthenate, to obtain a compound of theFormula IX: ##STR10## wherein X and B are as defined above.

The compound of the Formula IX is reacted with a methylene transferreagent, preferable methylene triphenylphosphorane, in a suitablesolvent, preferably tetrahydrofuran, to obtain a compound of the FormulaX: ##STR11## wherein X and B are as defined above.

The compound of the Formula X is reacted with a dihydroxylating agent,preferably osmium tetroxide, in the presence of a suitable oxidizingagent, preferably N-methylmorpholine-N-oxide, to obtain a compound ofthe Formula XI: ##STR12## wherein X and B are as defined above.

The compound of the Formula XI is converted to a compound of the FormulaI using any of the four processes described below.

In a first conversion process, the compound of the Formula XI is reactedwith a sulfonylating agent, preferably p-toluenesulfonyl chloride ormethanesulfonyl chloride, in the presence of a non-nucleophilic base,preferably triethylamine or diisopropylethyl amine, to give anintermediate mono-sulfonylated compound. This intermediate is thenreacted with a strong base, preferably sodium hydride, to obtain acompound of the Formula XII: ##STR13## wherein X and B are as definedabove.

The epoxide of Formula XII is reacted with a nitrogen containingnucleophile, preferably sodium azide, in the presence of a mildLewis-acid catalyst, preferably lithium perchlorate or magnesiumperchlorate, to obtain an intermediate alcohol azide. Reduction of thealcohol azide, preferably with hydrogen gas in the presence of a metalcatalyst, and subsequent protection with a suitable nitrogen-protectinggroup, preferably t-butoxycarbonyl, benzoxycarbonyl or benzyl, producesa compound of the Formula XIII: ##STR14## wherein X and B are as definedabove, and R₄ and R₅ are each independently hydrogen or a suitablenitrogen-protecting group. Preferred protecting groups aret-butoxycarbonyl, benzyloxycarbonyl and benzyl.

The compound of the Formula XIII is reacted with an acylating orsulfonylating agent, preferably methanesulfonyl chloride orp-toluenesulfonyl chloride, in the presence of a non-nucleophilic base,preferably triethylamine or diisopropylethylamine, in a suitable solventin which at least one of the reactants is at least partially soluble, toobtain an activated hydroxy group. The activated hydroxy group isdisplaced with a suitable nucleophile, preferably a thioacid salt, morepreferably potassium thioacetate, to obtain a compound of the FormulaXIV: ##STR15## wherein A, X, B, and R₄ and R₅ are as defined above, andAc is an acyl group. Preferably, Ac is acetyl.

Alternatively, the compound of the Formula XIII can be converted to thecompound of the Formula XIV in one chemical operation usingtriphenylphosphine, diethyl or dimethyl azadicarboxylate, and an acidicnucleophile, preferably thioacetic acid, in a suitable solvent.

The compound of the Formula XIV is treated with a nucleophilic base,preferably potassium carbonate, sodium carbonate, sodium hydroxide orpotassium hydroxide, in an alcoholic solvent, preferably methanol,ethanol or isopropanol, in the presence of an alkylating agent,preferably dimethyl or diethyl chloromalonate, to obtain a compound ofthe Formula XV: ##STR16## wherein A, X, B, and R₄ and R₅ are as definedabove, and each R₆ is independently hydrogen or a moiety that forms withthe attached CO₂ group a readily hydrolyzable ester group. Preferably,R₆ is C₁ -C₆ alkyl, hydroxyalkyl, alkylaryl or aralkyl. More preferably,R₆ is a C₁ -C₂ alkyl.

The compound of the Formula XV is treated under conditions suitable toremove either R₄ or R₅, or both protecting groups, to obtain a compoundof the Formula XVI: ##STR17## wherein A, X, B and R₆ are as definedabove. Where t-butoxycarbonyl is used as a protecting group, suitableconditions are treatment with trifluoroacetic acid, followed byneutralization.

The compound of the Formula XVI is reacted with an alkylating agent,preferably trimethyl or triethyl oxonium tetrafluoroborate, in asuitable solvent, preferably dichloromethane, to form an intermediatelactim ether. The intermediate lactim ether is reacted with guanidine inan alcoholic solvent, preferably methanol, ethanol or isopropanol, toform a compound of the Formula XVII: ##STR18## wherein A, X and B are asdefined above.

Alternatively, the compound of the Formula XVI can be converted to thecompound of the Formula XVII by reacting the compound of the Formula XVIwith a thiolating agent, preferably P₂ S₅ or2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide, toform the thiolactam intermediate. This intermediate is then alkylatedwith an alkylating agent, preferably methyl iodide or trimethyl ortriethyl oxonium tetrafluoroborate, and then with guanidine in analcoholic solvent, preferably methanol, ethanol or isopropanol, toobtain the compound of the Formula XVII.

Where B is an alcohol function--i.e., where the group attached with Bforms an ester group--the compound of the Formula XVII is hydrolyzedunder basic conditions to form a compound of the Formula XVIII:##STR19## wherein A and X are as defined above.

The compound of the Formula XVIII is peptide coupled, by means wellknown to those skilled in the art, with a glutamic acid diesterhydrochloride, to form a diester of the Formula XIX: ##STR20## whereinA, X, R₁ and R₂ are as defined above, except that neither R₁ nor R₂ ishydrogen.

Finally, if the free glutamic acid form is desired, the compound of theFormula XIX is hydrolyzed to form a compound of the Formula I.

In the second conversion process, a compound of the Formula XIV isprepared as described above. This compound is treated with acid,preferably trifluoroacetic, hydrochloric or p-toluenesulfonic acid, toremove all of the protecting groups (R₄, R₅ and Ac) to obtain a compoundof the Formula XX: ##STR21## wherein A, X and B are as defined above.

The compound of the Formula XX is reacted under weakly basic bufferconditions, preferably using a pH 7 phosphate buffer, in a suitablesolvent, preferably ethanol or methanol, with a compound having theFormula XXI: ##STR22## to obtain a compound of the Formula XVII. Theremainder of the second process, proceeding from the compound of theFormula XVII to a compound of the Formula I, is conducted in a manneranalogous to that described above.

In the third conversion process, the compound of the Formula XI isreacted with a suitable hydroxyl-protecting group, preferably atrialkylsilyl group, more preferably a t-butyldimethylsilyl chloride, inthe presence of a mild non-nucleophilic base, preferably triethylamine,to obtain a compound of the Formula XXII: ##STR23## wherein X and B areas defined above, and R₇ is a suitable hydroxyl-protecting group,preferably a trialkylsilyl group.

The compound of the Formula XXII is then reacted with an acylating orsulfonylating agent, preferably methansulfonyl chloride orp-toluenesulfonyl chloride, in the presence of a non-nucleophilic base,preferably triethylamine or diisopropylethylamine, in a suitable solventin which at least one of the reactants is at least partially soluble, toobtain an activated hydroxy group. The activated hydroxy group isdisplaced with a suitable nucleophile, preferably a thioacid salt, morepreferably potassium thioacetate, to obtain a compound of the FormulaXXIII: ##STR24## wherein A, X, B, R₇ and Ac are as defined above.

Alternatively, the compound of the Formula XXII can be converted to thecompound of the Formula XXIII in one chemical operation usingtriphenylphosphine or diethyl or dimethyl azadicarboxylate, and anacidic nucleophile, preferably thioacetic acid, in a suitable solvent.

The compound of the Formula XXIII is reacted with a nucleophilic base ora mild acid to selectively remove the acyl group on moiety A. Theresulting intermediate is reacted with a compound of the Formula XXIV:##STR25## in the presence of a non-nucleophilic base, preferablytriethylamine, diisopropylethylamine or potassium carbonate, to obtain acompound of the Formula XXV: ##STR26## wherein A, X, B and R₇ are asdefined above.

The protecting group R₇ on the compound of the Formula XXV is removed bytreatment with a suitable reagent to obtain a compound of the FormulaXXVI: ##STR27## wherein A, X and B are as defined above. Where R₇ istrialkylsilyl, the reagent is preferably a fluoride salt, morepreferably potassium fluoride, tetrabutylammonium fluoride or cesiumfluoride.

The compound of the Formula XXVI is cyclized to obtain the compound ofthe Formula XVII by activating the hydroxy group with an activatingagent, preferably methanesulfonyl chloride, followed by treatment with abase. Alternatively, the nitrogen of the pyrimidinone is first protectedwith a suitable protecting group, preferably t-butoxycarbonyl, followedby cyclization and subsequent removal of the protecting group underacidic conditions. The remainder of the process proceeds from thecompound of the Formula XVII to a compound of the Formula I in a manneranalogous to that described above.

In the fourth and preferred conversion process, an alcohol compound ofthe Formula XXVI is prepared as described above. This alcohol is reactedwith a suitable oxidizing agent to produce an aldehyde functionalitythat cyclizes to the compound of the Formula XXVII: ##STR28## wherein A,X and B are as defined above.

The compound of the Formula XXVII is reacted with a reducing agent,preferably sodium cyanoborohydride, in the presence of a Lewis acid,preferably boron trifluoride etherate, to obtain a compound of theFormula XVII defined above. The rest of the process proceeds from thecompound of the Formula XVII to a compound of the Formula I in a manneranalogous to that described above.

The compounds of the Formula I where Z is other than CH₂ can be preparedin an analogous manner to those where Z is CH₂. In particular, compoundsof the Formula I wherein Z is other than CH₂ can be prepared using anolefin of the Formula XXXIV: ##STR29## wherein X and R₆ are as definedabove, and Z is as defined above for Formula I except that it is otherthan CH₂.

Where Z is sulfur, oxygen, or a substituted or unsubstituted amino, acompound of the Formula XXXV: ##STR30## wherein X and R₆ are as definedabove, and Z is sulfur, oxygen, or a substituted or unsubstituted amino,is alkylated. The alkylation can be accomplished using an allylhalide,preferably allylbromide, in the presence of a non-nucleophilic base,preferably triethylamine or diisopropylethylamine, to obtain thecompound of the Formula XXXIV.

Where Z is a substituted or unsubstituted C₁ -C₂ alkyl other than CH₂, asubstituted or unsubstituted C₂ -C₃ alkenyl or a substituted orunsubstituted C₂ -C₃ alkynyl, the compound of the Formula XXXIV isprepared by olefination of an aldehyde of the Formula XXXVI: ##STR31##wherein X and R₆ are as defined above, and Z is a substituted orunsubstituted C₁ -C₂ alkyl other than CH₂, a substituted orunsubstituted C₂ -C₃ alkenyl or a substituted or unsubstituted C₂ -C₃alkynyl. The aldehyde of the Formula XXXVI can be prepared in a manneranalogous to that described by Chuan Shih et al., Journal of MedicinalChemistry, vol. 35 (1992), 1109-1116. The olefination of the aldehydecan be accomplished using a methylene transfer agent, preferablymethylene-triphenylphosphorane.

The compound of the Formula XXXIV is reacted with a dihydroxylatingagent, preferably osmium tetroxide, in the presence of a suitableoxidizing agent, preferably N-methylmorpholine-N-oxide, to obtain acompound of the Formula XXXVII: ##STR32## wherein X and R₆ are asdefined above; and Z is as defined above for Formula I, except that itis other than CH₂.

The compound of the Formula XXXVII is reacted with a sulfonylatingagent, preferably p-toluenesulfonyl chloride or methanesulfonylchloride, in the presence of a non-nucleophilic base, preferablytriethylamine or diisopropylethylamine, to yield an intermediatemono-sulfonylated compound. This intermediate is reacted with a strongbase, preferably sodium hydride, to produce a compound of the FormulaXXXVIII: ##STR33## wherein X and R₆ are as defined above, and Z is asdefined for Formula I except that it is other than CH₂.

The epoxide of Formula XXXVIII is reacted with a nitrogen-containingnucleophile, preferably sodium azide, in the presence of a mildLewis-acid catalyst, preferably lithium or magnesium perchlorate, to anobtain an intermediate alcohol azide. This intermediate is reduced,preferably with hydrogen gas in the presence of a metal catalyst, andsubsequent protection with a suitable nitrogen-protecting group,preferably t-butoxycarbonyl, benzoxycarbonyl or benzyl, to produce acompound of the Formula XVII': ##STR34## wherein X, R₆, and R₄ and R₅are as defined above, and Z is as defined for Formula I except that itis other than CH₂.

The compound of the Formula XVII' is then reacted with an acylating orsulfonylating agent, preferably methanesulfonyl chloride orp-toluenesulfonyl chloride, in the presence of a non-nucleophilic base,preferably triethylamine or diisopropylethylamine, in a suitable solventin which at least one of the reactants is at least partially soluble, toobtain an activated hydroxy group. The activated hydroxy group isdisplaced with a suitable nucleophile, preferably a thioacid salt, morepreferably potassium thioacetate, to obtain a compound of the FormulaXVIII': ##STR35## wherein A, X, R₆, R₄ and R₅, and Ac are as definedabove, and Z is as defined for Formula I except that it is other thanCH₂.

Alternatively, the compound of Formula XVII' is converted to thecompound of Formula XVIII' in one chemical operation usingtriphenylphosphine, diethyl or dimethyl aza-dicarboxylate, and an acidicnucleophile, preferably thioacetic acid, in a suitable solvent.

The compound of the Formula XVIII' is treated with a nucleophilic base,preferably potassium carbonate, sodium carbonate, sodium hydroxide orpotassium hydroxide, in an alcoholic solvent, preferably methanol,ethanol or isopropanol, in the presence of an alkylating agent,preferably dimethyl or diethyl chloromalonate, to obtain a compound ofthe Formula XIX': ##STR36## wherein A, X, R₆, and R₄ and R₅ are asdefined above, and Z is as defined for Formula I except that it is otherthan CH₂.

The compound of the Formula XIX' is treated under conditions suitable toremove either or both of the R₄ and R₅ protecting groups to produce acompound of the Formula XX': ##STR37## wherein A, X and R₆ are asdefined above, and Z is as defined for Formula I except that it is otherthan CH₂. Where t-butoxycarbonyl is a protecting group, the conditionsfor removal of this group are preferably treatment with trifluoroaceticacid followed by neutralization to produce the compound of the FormulaXX'.

The compound of the Formula XX' is reacted with an alkylating agent,preferably trimethyl or triethyl oxonium tetrafluoroborate, in asuitable solvent, preferably dichloromethane, to form an intermediatelactim ether. The intermediate lactim ether is reacted with guanidine inan alcoholic solvent, preferably methanol, ethanol or isopropanol, toform a compound of the Formula XXI': ##STR38## wherein A, X and R₆ areas defined above, and Z is as defined for Formula I except that it isother than CH₂.

Alternatively, the compound of the Formula XX' is converted to thecompound of the Formula XXI' by reacting the compound of the Formula X'with a thiolating agent, preferably P₂ S₅ or2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide toform the thiolactam intermediate. This can then be alkylated with analkylating agent, preferably methyl iodide or trimethyl or triethyloxonium tetrafluoroborate, and then with guanidine in an alcoholicsolvent, preferably methanol, ethanol or isopropanol, to obtain thecompound of the Formula XXI'.

The compound of the Formula XXI' is hydrolyzed under basic conditions toform a compound of the Formula XXII': ##STR39## wherein A and X are asdefined above, and Z is as defined for Formula I except that it is otherthan CH₂. Where R₆ is hydrogen in the compound of the Formula XXI', thenthe hydrolyzation reaction is not necessary, and the compound of theFormula XXI' is peptide coupled as described below.

The compound of the Formula XXII' (or the compound of the Formula XXI'where R₆ is hydrogen), which is in the free carboxylic acid form, can bepeptide coupled, by means well known to those skilled in the art, with aglutamic acid diester hydrochloride to form a diester of the FormulaXXIII': ##STR40## wherein A, X and are as defined for Formula XXII', andR₁ and R₂ are each independently a moiety that forms with the attachedCO₂ a readily hydrolyzable ester group, such as a C₁ -C₆ alkyl,hydroxyalkyl, alkylaryl or arylalkyl.

Finally, if the free acid form is desired, the compound of the FormulaXXIII' is hydrolyzed to produce compounds of the Formula I where R₁ andR₂ are each H.

A detailed example of the preparation of a compound of the Formula I isprovided below.

EXAMPLE 1

N-(5-[2-(2-amino-4(3H)-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-6-yl)ethyl]-4-methylthieno-2-yl)-L-glutamicacid (Compound 1) ##STR41## Synthesis

Compound 1 was synthesized by the following process.

a. 5-bromo-4-methylthiophene-2-carboxylic acid: ##STR42##

This compound was prepared according to M. Nemec, Collection Czechoslov.Chem. Commun., vol. 39 (1974), 3527.

b. 6-ethynyl-2-(pivaloylamino)-4(3H)-oxopyrido [2,3-d]pyrimidine:##STR43##

This compound was prepared according to E. C. Taylor & G. S. K. Wong, J.Org. Chem., vol. 54 (1989), 3618.

c. Diethyl N-(5-bromo-4-methylthieno-2-yl)-L-glutamate: ##STR44##

To a stirred solution of 5-bromo-4-methylthiophene-2-carboxylic acid(3.32 g, 15 mmol), 1-hydroxybenzotriazole (2.24 g, 16.6 mmol),L-glutamic acid diethyl ester hydrochloride (3.98 g, 16.6 mmol) anddiisopropylethylamine (2.9 ml, 2.15 g, 16.6 mmol) in dimethylformamide(DMF) (40 ml) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (3.18 g, 16.6 mmol). The resulting solution was stirredunder argon at ambient temperature for 18 hours, poured into brine (300ml), diluted with water (100 ml) and extracted with ether (3×120 ml).The combined organic extracts were washed with water (150 ml), driedover MgSO₄ and concentrated in vacuo to give a brown gum, which waspurified by flash chromatography. Elution with hexane: EtOAc (2:1)provided the product as an orange oil (5.05 g, 83% yield). Analysesindicated that the product was diethyl N-(5-bromo-4-methylthieno-2-yl)glutamate. NMR(CDCl₃) δ: 7.22 (1H, s), 6.86 (1H, d, J=7.5 Hz), 4.69 (1H,ddd, J=4.8, 7.5, 9.4 Hz), 4.23 (2H, q, J=7.1 Hz), 4.12 (2H, q, J=7.1Hz), 2.55-2.39 (2H, m), 2.35-2.22 (1H, m), 2.19 (3H, s), 2.17-2.04 (1H,m), 1.29 (3H, t, J=7.1 Hz), 1.23 (3H, t, J=7.1 Hz).

Anal. (C₁₅ H₂₀ NO₅ SBr) C,H,N,S,Br.

d. diethyl N-(5-[(2-[pivaloylamino]-4(3H)-oxopyrido[2,3-d]pyrimidin-6-yl) ethynyl]-4-methylthieno-2-yl) glutamate:##STR45##

To a stirred solution of diethyl N-(5-bromo-4-methylthieno-2-yl)glutamate (4.21 g, 10.4 mmol) in acetonitrile (55 ml) under an argonatmosphere were added bis (triphenylphosphine) palladium chloride (702mg, 1.0 mmol), cuprous iodide (200 mg, 1.1 mmol), triethylamine (1.5 ml,1.09 g, 10.8 mmol) and6-ethynyl-2-(pivaloylamino)-4(3H)-oxopyrido[2,3-d]pyrimidine (5.68 g, 21mmol). The resultant suspension was heated at reflux for 6 hours. Aftercooling to room temperature, the crude reaction mixture was filtered andthe precipitate was washed with acetonitrile (50 ml) and ethylacetate(EtOAc) (2×50 ml). The combined filtrates were concentrated in vacuo togive a brown resin, which was purified by flash chromatography. Elutionwith CH₂ Cl₁₂ : CH₃ OH (49:1) provided the product as an orange solid(4.16 g, 67% yield). Analyses indicated that the product was diethylN-(5-[(2-[pivaloylamino]-4(3H)-oxopyrido[2,3-d]pyrimidin-6-yl)ethynyl]-4-methylthieno-2-yl) glutamate. NMR (CDCl₃) δ: 8.95 (1H, d,J=2.2 Hz), 8.59 (1H, d, J=2.2 Hz), 7.33 (1H, s), 7.03 (1H, d, J=7.4 Hz),4.73 (1H, ddd, J=4.8, 7.4, 9.5 Hz), 4.24 (2H, q, J=7.1 Hz), 4.13 (2H, q,J=7.1 Hz), 2.55-2.41 (2H, m), 2.38 (3H, s), 2.35-2.24 (1H, m), 2.19-2.05(1H, m), 1.34 (9H, s), 1.30 (3H, t, J=7.1 Hz), 1.24 (3H, t, J=7.1 Hz).

Anal. (C₂₉ H₃₃ N₅ O₇ S.0.75H₂ O) C,H,N,S.

e. diethyl N-(5-[(2-[pivaloylamino]-4(3H)-oxopyrido[2,3,d]pyrimidin-6-yl)ethyl]-4-methylthieno-2-yl) glutamate: ##STR46##

A suspension of diethylN-(5-[(2-[pivaloylamino]-4(3H)-oxopyrido[2,3-d]pyrimidin-6-yl)ethyl]-4-methylthieno-2-yl)glutamate (959 mg, 1.6 mmol) and 10% Pd on carbon (1.5 g, 150% wt. eq.)in trifluoroacetic acid (30 ml) was shaken under 50 psi of H₂ for 22hours. The crude reaction mixture was diluted with CH₂ C₂, filteredthrough a pad of Celite (diatomaceous earth) and concentrated in vacuo.The residue obtained was dissolved in CH₂ Cl₂ (120 ml), washed withsaturated NaHCO₃ (2×100 ml), dried over Na₂ SO₄ and concentrated invacuo to give a brown gum, which was purified by flash chromatography.Elution with CH₂ Cl₂ :CH₃ OH (49:1) provided the product as a yellowsolid (772 mg, 80% yield). Analyses indicated that the product wasdiethylN-(5-[(2-[pivaloylamino]-4(3H)-oxopyrido[2,3-d]pyrimidin-6-yl)ethyl]-4-methylthieno-2-yl)glutamate. NMR (CDCl₃) δ: 8.60 (1H, d, J=2.2 Hz), 8.49 (1H, broad), 8.32(1H, d, J=2.2 Hz), 7.22 (1H, s), 6.78 (1H, d, J=7.5 Hz), 4.72 (1H, ddd,J=4.8, 7.5, 9.5 Hz), 4.23 (2H, q, J=7.1 Hz), 4.11 (2H, q, J=7.1 Hz),3.12-3.00 (4H, m), 2.52-2.41 (2H, m), 2.37-2.22 (1H, m), 2.16-2.04 (1H,m), 2.02 (3H, s), 1.33 (9H, s), 1.29 (3H, t, J=7.1 Hz), 1.23 (3H, t,J=7.1 Hz). Anal. (C₂₉ H₃₇ N₅ O₇ S.0.5H₂ O) C,H,N,S.

f. diethylN-(5-[(2-[pivaloylamino]-4(3H)-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-6-yl)ethyl]-4-methylthieno-2-yl)glutamate: ##STR47##

A suspension of diethylN-(5-[(2-[pivaloylamino]-4(3H)-oxopyrido[2,3-d]pyrimidin-6-yl)ethyl]-4-methylthieno-2-yl)glutamate (2.98 g, 5 mmol), 10% Pt on carbon (1.5 g, 50% wt. eq.) andPtO₂ (1.5 g, 50% wt. eq.) in trifluoroacetic acid (170 ml) was shakenunder 800 psi of H₂ for 40 hours. The crude reaction mixture was dilutedwith CH₂ Cl₂, filtered through a pad of Celite, and concentrated invacuo. The residue obtained was dissolved in CH₂ Cl₂ (150 ml), washedwith saturated NaHCO₃ (2×150 ml), dried over Na₂ SO₄, and concentratedin vacuo to give a brown resin, which was purified by flashchromatography. Elution with CH₂ Cl₂ ; CH₃ OH (24:1) provided initiallyan unreacted substrate (1.42 g, 48% yield) and then the product as ayellow solid (293 mg, 10% yield). Analyses indicated that the productwas diethylN-(5-[(2-[pivaloylamino]-4(3H)-oxo-5,6,7,8-tetrahydropyrido-[2,3-d]pyrimidin-6-yl)ethyl]-4-methylthieno-2-yl)glutamate. NMR (CDCl₃) δ: 7.24 (1H, s), 6.75 (1H, d, J=7.6 Hz), 5.57(1H, broad) , 4.72 (1H, ddd, J=4.8, 7.6, 12.6 Hz), 4.22 (2H, q, J=7.1Hz), 4.11 (2H, q, J=7.1 Hz), 3.43-3.36 (1H, m), 3.06-2.98 (1H, m),2.89-2.68 (3H, m), 2.52-2.40 (3H, m), 2.37-2.23 (1H, m), 2.15 (3H, s),2.14-2.03 (1H, m), 1.94-1.83 (1H, m), 1.73-1.63 (2H, m), 1.32 (9H,s),1.29 (3H, t, J=7.1 Hz), 1.23 (3H, t, J=7.1 Hz). Anal. (C₂₉ H₄₁ N₅ O₇S.0.5H₂ O) C,H,N,S.

g.N-(5-[2-(2-amino-4(3H)-oxo-5,6,7,8-tetrahydropyrido-[2,3-d]pyrimidin-6-yl)ethyl]-4-methylthieno-2-yl)glutamic acid (Compound 1):

A solution of diethylN-(5-[(2-[pivaloylamino]-4(3H)-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-6-yl)ethyl]-4-methylthieno-2-yl)glutamate (293 mg, 0.5 mmol) in 1N NaOH (25 ml) was stirred at ambienttemperature for 90 hours, then neutralized with 6N HCl. The precipitatethat formed was collected by filtration and washed with water (4×10 ml)to provide the product as a yellow solid (63 mg, 28% yield). Analysesindicated that the product wasN-(5-[2-(2-amino-4(3H)-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-6-yl)ethyl]-4-methylthieno-2-yl)glutamic acid. NMR (DMSO-d6) δ: 12.44 (2H, broad), 9.89 (1H, broad),8.42 (1H, d, J=7.8 Hz), 7.57 (1H, s), 6.39 (1H, br s), 6.12 (2H, br s),4.30 (1H, ddd, J=4.8, 7.8, 9.6 Hz), 3.26-3.18 (2H, m), 2.83-2.74 (3H,m), 2.31 (2H, t, J=7.4 Hz), 2.12 (3H, s), 2.09-2.01 (1H, m), 1.94-1.80(2H, m), 1.68-1.47 (3H,m). Anal. (C₂₀ H₂₅ N₅ O₆ S.1.1H₂ O) C,H,N,S.

Biological and Biochemical Evaluation

Determination of Inhibition Constants for GAR Transformylase:

The GAR-transformylase (GARFT) assay method of Young et al.,Biochemistry 23 (1984), 3979-3986, was modified and used as describedbelow. Reactions mixtures contained the catalytic domain of the humanGARFT, 0-250 nM of the test compound, 20 μM glycinamide ribonucleotide(GAR), 10 or 20 μM N¹⁰ -formyl-5,8-dideazafolate (FDDF), 50 mM HEPES-KOH(pH 7.5), and 50 mM KCl. The reaction was initiated with the addition ofenzyme to a final concentration of 11 nM, followed by monitoring of theincrease in absorbance at 294 nm at 20° C. (e₂₉₄ =18.9 mM⁻¹ cm⁻¹).

The GARFT inhibition constant (K_(i)) was determined from the dependenceof the steady-state catalytic rate on inhibitor and substrateconcentration. The type of inhibition observed was determined to becompetitive with respect to FDDF by the dependence of the apparent K_(i)(K_(i),app) on the concentration of FDDF and was shown to be describedby K_(i),app =K_(i) +(K_(i) /K_(m))[FDDF]. The Michaelis constant forFDDF, K_(m), was determined independently by the dependence of thecatalytic rate on FDDF concentration. Data for both the K_(m) and K_(i)determinations were fitted by non-linear methods to the Michaelisequation, or to the Michaelis equation for competitive inhibition, asappropriate. Data resulting from tight-binding inhibition was analyzedand K_(i) was determined by fitting the data to the tight-bindingequation of Morrison, Blochem Biophys Acta 185 (1969), 269-286, bynonlinear methods.

Determination of Dissociation Constants for Human Folate BindingProtein:

The dissociation constant (Kd) for human folate-binding protein (FBP)was determined in a competitive binding assay using membrane associatedFBP prepared from cultured KB cells. Preparation of KB cell MembraneFraction:

Adherent KB cells were scraped from flasks, washed once in ice-cold PBS,and centrifuged at 5000× g for 5 minutes at 4° C. Pelleted cells (2×10⁸cells) were resuspended in 10 ml of suspension buffer (KH₂ PO₄ -KOH pH7.4:10 mM EDTA: 10 mM 2-mercaptoethanol), sonicated briefly to completecell lysis and centrifuged at 12000× g for 10 minutes at 4° C. Thepellet was stripped of endogenous bound folate by resuspension in 20 mlof acidic buffer (50 mM KH₂ PO₄ -KOH pH 3.5:10 mM EDTA: 10 mM2-mercaptoethanol) and centrifuged as before. The pellet was thenresuspended in 20 ml of the suspension buffer at pH 7.4 and centrifugedas before. The pellet was resuspended in 5 ml of suspension buffer at pH7.4 lacking EDTA. Protein content was quantitated using the Bradfordmethod with BSA as standard. Typical yields for this procedure were 4-5mg total membrane protein per 2×10⁸ cells. This final suspension wasused as a source of membrane-associated human FBP. FBP CompetitiveBinding Assay:

Inhibitor was allowed to compete against ³ H-folic acid for binding toFBP. Reactions mixtures contained 50-100 mg of cell membrane proteincontaining 3-6 pmoles (3-6 nM) of FBP, 17.25 pmoles ³ H-folic acid(17.25 nM, 0.5 μCi), various concentrations of competitor, in 1 ml of 50mM KH₂ PO4-KOH pH 7.4 : 10 mM 2-mercaptoethanol. Reactions wereperformed at 25° C. Because of the very slow release of bound ³ H-folicacid, the competitor was prebound for 30 minutes in the absence of ³H-folic acid. ³ H-Folic acid was then added and the mixtures wereallowed to equilibrate for 2.5 hours. The full reaction mixtures weredrawn through nitrocellulose filters under vacuum to trap the cellmembranes with bound ³ H-folic acid. The trapped membranes were thenwashed 4 times with 1 ml of reaction buffer. The amount of bound ³H-folic acid was measured by scintillation counting of thenitrocellulose membrane. The data obtained were nonlinearly fitted asdescribed above. The FBP K_(d) for ³ H-folic acid, used to calculate thecompetitor K_(d), was obtained by direct titration of FBP with ³H-folate and subsequent nonlinear fitting of the data to a tight-bindingK_(d) equation.

Cell lines:

The cell lines used and their origin are tabulated in Table 1. Thegrowth conditions and media requirements of each cell line aresummarized in Table 2. All cultures were maintained at 37° C., 5%air-CO₂ in a humidified incubator. In vitro growth inhibition:

Stock solutions of the inhibitors were prepared in 10 mM sodiumbicarbonate in water and stored in 1 ml aliquots at -20° C. for cellculture experiments. Cell-growth inhibition was measured by amodification of the method of Mosmann, J. Immunol. Methods 65 (1983),55-63.

Mid-log phase cells of each cell line were diluted to 18,500 cells/ml infresh RPMI growth medium (Mediatech, Washington, D.C.) supplemented withdialyzed fetal-calf serum (Hyclone Laboratories Inc., Logan, Utah), andthen aliquotted into columns 2 through 12 of 96-well microtiter plates.Column 1 was filled with the same volume, 135 ml, of fresh medium,without cells, for use as a blank. The plates were then placed in a 37°C., 5% air-CO₂ incubator. After 1 to 4 hours, plates were removed fromthe incubator followed by addition of the test compound at 10× finalconcentration, 15 ml/well in binary dilutions, to columns 12 to 4. Forreversal experiments, hypoxanthine (1.75 mM) or AICA (1.75 mM) wasincluded in all drug solutions (final concentration 175 mM). Wellscontaining each concentration of test compound were prepared inquadruplicate on each plate. Fifteen milliliters of media, without testcompound, were added to the wells in column 1 of the plates. The cellswere then returned to the incubator and remained undisturbed for thefull incubation period. On day 3 for L1210 and L1210/CI920 cells or day5 for CCRF-CEM cells, 50 ml of 0.8 mg/ml MTT(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigmacatalog no. M2128) dissolved in tissue culture medium was added to eachwell of all plates, after which cells were returned to the incubator.After 4 hours, all plates were removed from the incubator andcentrifuged at 1200 rpm for 7 minutes. Media were siphoned off and 150ml of DMSO was added to each well of all plates. Plates were then mixedat slow speed on a vortex mixer for 1 hour in the dark at roomtemperature. The extent of metabolized MTT was measuredspectrophotometrically at 540 nm on a Molecular Devices Vmax™ kineticmicroplate reader. The concentration of drug required to reduce cellgrowth by 50% as measured by MTT metabolism was determined byinterpolation between the O.D. (minus blank) immediately above and below50% of control O.D. (minus blank).

                  TABLE 1                                                         ______________________________________                                        Tissue of Origin and Source of Cell                                           Lines Employed in In Vitro Studies                                            Cell Line                                                                              Source     Origin                                                    ______________________________________                                        L1210    ATCC.sup.# Mouse, lymphocytic leukemia                               CCRF-CEM ATCC.sup.# Human, acute lymphoblastic                                                    leukemia                                                  ______________________________________                                         .sup.# ATCC = American Type Culture Collection                           

                  TABLE 2                                                         ______________________________________                                        Culture Conditions, Plating Densities and                                     Incubation Times Used in Microtiter Assays                                                        DFCS    Plating  Incubation                               Cell                Conc.*  Density  Time                                     line     Medium     (%)     (cells/well)                                                                           (days)                                   ______________________________________                                        L1210    RPMI-1640  5       2500     3                                        CCRF-CEM RPMI-1640  10      2500     5                                        ______________________________________                                         *DFCS Conc. = dialyzed fetal calf serum concentration.                   

                  TABLE 3                                                         ______________________________________                                        Comparative Data for Test Compound and 6R-DDATHF                              Growth Inhibition Using Continuous (72-hour) Exposure                                          IC.sub.50 Cell                                                                         IC.sub.50 Cell                                                                         Human Folate                                                Culture  Culture  Binding                                            GARFT    L1210    CCRF-CEM Protein                                    Compound                                                                              K.sub.i (nM)                                                                           (nM).sup.a                                                                             (nM).sup.a                                                                             K.sub.d (nM)                               ______________________________________                                        1       1.4      13.5     6.1      28                                         DDATHF.sup.b                                                                          25       17.5     1.5      0.020                                      ______________________________________                                         .sup.a : Mean IC.sub.50 ± standard deviation;                              .sup.b : 6RDDATHF, the 6R diastereomer of 5,,10dideazatetrahydrofolic aci     (Lometrexol) (See F.M. Muggia, "Folate antimetabolites inhibitor to de        novo purine synthesis," New Drugs, Concepts and Results in Cancer             Chemotherapy, Kluwer Academic Publishers, Boston (1992), 65-87.          

As the above comparative data show, Compound 1 has a relative folatebinding protein K_(d) that is about 1400 times less potent than6R-DDATHF.

EXAMPLE 2

N-(5-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-6]-[1,4]thiazin-6-yl)ethyl]-4-methylthieno-2-yl)-L-glutamic acid (Compound 2)##STR48## Compound 2 was prepared as follows.

a. methyl 5-bromo-4-methylthiophene-2-carboxylate: ##STR49##

To a solution of 5-bromo-4-methylthiophene-2-carboxylic acid (20.32 g,92 mmol) in CH₃ OH (450 ml) was added concentrated H₂ SO₄ (4 ml). Theresultant solution was heated at reflux for 18 hours. The solvent wasremoved by concentration in vacuo, and the residue obtained waspartitioned between saturated NaHCO₃ (350 ml) and ether (350 ml). Thelayers were separated and the aqueous phase extracted with ether (3×150ml). The combined organic extracts were dried over MgSO₄ andconcentrated in vacuo to give a red oil, which was purified by flashchromatography. Elution with hexane:ethyl acetate (9:1) provided theproduct as a yellow oil, which solidified on standing (18.34 g, 85%yield). Analyses indicated that the product was methyl5-bromo-4-methylthiophene-2-carboxylate. NMR (CDCl₃) δ: 7.47 (1H, s),3.86 (³ H, s), 2.20 (³ H, s). Anal. (C₇ H₇ O₂ SBr) C,H,S,Br.

b. methyl 5-(3-hydroxypropynyl)-4-methylthiophene-2-carboxylate:##STR50##

To a stirred solution of methyl 5-bromo-4-methylthiophene-2-carboxylate(5.18 g, 22 mmol) in diethylamine (60 ml) under an argon atmosphere wereadded bis(triphenylphosphine) palladium chloride (77 mg, 0.11 mmol),cuprous iodide (42 mg, 0.22 mmol) and propargyl alcohol (1.5 ml, 1.44 g,26 mmol). The resultant mixture was stirred at ambient temperature for18 hours. The solvent was removed by concentration in vacuo, and theresidue obtained was diluted with water (200 ml) and then extracted withEtOAc (3×100 ml). The combined organic extracts were washed with 0.5 NHCl (100 ml), dried over MgSO₄ and concentrated in vacuo to give a brownoil, which was purified by flash chromatography. Elution withhexane:EtOAc (2:1) provided the product as an orange oil, whichsolidified on standing (4.07 g, 88% yield). Analyses indicated that theproduct was methyl5-(3-hydroxypropynyl)-4-methylthiophene-2-carboxylate. NMR (CDCl₃) δ:7.52 (1H, s), 4.55 (2H, s), 3.87 (3H, s), 2.29 (3H, s). Anal. (C₁₀ H₁₀O₃ S) C,H,S.

c. methyl 5-(3-hydroxypropyl)-4-methylthiophene-2-carboxylate: ##STR51##

A suspension of methyl5-(3-hydroxypropynyl)-4-methylthiophene-2-carboxylate (3.86 g, 18 mmol)and 5% Pd on carbon (0.72 g, 19% wt. eq.) in EtOAc (110 ml) was shakenunder 50 psi of H₂ for 20 hours. The crude reaction mixture was filteredthrough a pad of Celite, and the filtrate was concentrated in vacuo toprovide the product as a yellow oil (3.84 g, 98% yield). Analysesindicated that the product was methyl5-(3-hydroxypropyl)-4-methylthiophene-2-carboxylate. NMR (CDCl₅) δ: 7.51(1H, s), 3.84 (3H, s), 3.71 (2H, t, J=6.2 Hz), 2.86 (2H, t, J=7.6 Hz),2.16 (3H, s), 1.92 (2H, tt, J=6.2, 7.6 Hz). Anal. (C₁₀ H₁₄ O₃ S) C,H,S.

d. methyl 4-methyl-5-(3-oxopropyl) thiophene-2-carboxylate: ##STR52##

To a stirred suspension of methyl5-(3-hydroxypropyl)-4-methylthiophene-2-carboxylate (3.74 g, 17 mmol),N-methylmorpholine-N-oxide (3.00 g, 26 mmol) and powdered 4Å molecularsieves (4.5 g) in CH₂ Cl₂ (50 ml) was added tetrapropylammoniumperruthenate (300 mg, 0.85 mmol). The resultant suspension was stirredat ambient temperature for 40 minutes. The solvent was removed byconcentration in vacuo, and the residue obtained was purified by flashchromatography. Elution with hexane:EtOAc (4:1) provided the product asa yellow oil (1.82 g, 49% yield) Analyses indicated that the product wasmethyl 4-methyl-5-(3-oxopropyl) thiophene-2-carboxylate. NMR (CDCl₃) δ:9.83 (1H, t, J=0.8 Hz), 7.50 (1H, s), 3.84 (3H, s), 3.07 (2H, t, J=7.4Hz), 2.83 (2H, dt, J=0.8, 7.4 Hz), 2.17 (3H, s). Anal. (C₁₀ H₁₂ O₃ S)C,H,S

e. methyl 5-(3-butenyl)-4-methylthiophene-2-carboxylate: ##STR53##

To a stirred suspension of methyltriphenylphosphonium bromide (3.14 g,8.8 mmol) in THF (30 ml) under an argon atmosphere at 0° C. was added2.5M n-butyllithium in hexane (3.4 ml, 8.5 mmol). The resultant slurrywas stirred for 10 minutes at 0° C., for 75 minutes at ambienttemperature, and then cooled to -65° C. prior to the dropwise additionof a solution of the methyl 4-methyl-5(3-oxopropyl)thiophene-2-carboxylate (1.71 g, 8.1 mmol) in THF (30 ml). The coolingbath was removed and the reaction was stirred for 90 minutes whilegradually warming to room temperature. The crude reaction mixture wasconcentrated in vacuo to a volume of 20 ml, diluted with ether (200 ml),and filtered through a pad of celite. The filtrate was concentrated invacuo to give an orange oil, which was purified by flash chromatography.Elution with hexane:EtOAc (95:5) provided the product as a yellow oil(772 mg, 46%). Analyses indicated that the product was methyl5-(3-butenyl)-4-methylthiophene-2-carboxylate. NMR (CDCl₃) δ: 7.50 (1H,s), 5.84 (1H, ddt, J=10.2, 17.0, 6.6 Hz), 5.07 (1H, dd, J=1.6, 17.0 Hz),5.02 (1H, dd, J=1.6, 10.2 Hz), 3.84 (³ H, s). Anal. (C₁₁ H₁₄ O₂) C,H,S.

f. methyl 5-(3,4-dihydroxybutyl)-4-methylthiophene-2-carboxylate:##STR54##

To a stirred solution of N-methylmorpholine-N-oxide (735 mg, 6.3 mmol)and osmium tetroxide (5 mg, 0.02 mmol) in acetone (30 ml) was added asolution of methyl 5-(3-butenyl)-4-methylthiophene-2-carboxylate (701mg, 3.3 mmol) in acetone (20 ml). The resultant solution was stirredunder an argon atmosphere at ambient temperature for 48 hours, thenfiltered through a pad of Celite. The filtrate was acidified by additionof 0.5M H₂ SO₄ (10 ml), and the acetone was removed by concentration invacuo. The aqueous residue was diluted with water (20 ml) and extractedwith EtOAc (3×25 ml). The combined organic extracts were washed withwater (3×25 ml), dried over Na₂ SO₄, and concentrated in vacuo to give abrown gum, which was purified by flash chromatography. Elution with CH₂Cl₂ ; EtOAc (2:3) provided the product as an off-white solid (577 mg,71% yield). Analyses indicated that the product was methyl5-(3,4-dihydroxybutyl)-4-methylthiophene-2-carboxylate. NMR (CDCl₃) δ:7.50 (1H, s), 3.84 (³ H, s), 3.79-3.72 (1H, m), 3.86 (1H, dd, J=3.2,10.9 Hz), 3.48 (1H, dd, J=7.4, 10.9 Hz), 3.00-2.80 (2H, m). Anal. (C₁₁H₁₆ O₄ S) C,H,S.

The above examples are given to illustrate various aspects of theinvention. It is to be understood that appropriate modifications will bewithin the capabilities of one having ordinary skill in the art in lightof the teachings contained herein.

Where possible as a matter of chemistry, chemical groups recited hereincan be substituted. In some cases, this possibility is made explicit byreciting, e.g., substituted or unsubstituted C₁ -C₃ alkyl group.

Where more than one R₆ group is recited in any Formula herein, each R₆can be independently selected from the possibilities given.

What is claimed is:
 1. A compound of the formula II: ##STR55## wherein:R is a halogen;X is a substituted or unsubstituted C₁ -C₆ alkyl group;and B is glutamic acid or a derivative thereof, linked through the aminoportion to form an amide.
 2. A compound according to claim 1, wherein Ris bromo.
 3. A compound according to claim 1, wherein when X issubstituted, the substituents are selected from OH, NH₂, O-methyl,O-ethyl, SH, SCH₃ and NH-methyl.
 4. A compound according to claim 1,wherein X is unsubstituted.
 5. A compound according to claim 4, whereinX is methyl or ethyl.
 6. A compound according to claim 1, wherein B isdiethyl glutamate.
 7. A compound of the formula VII: ##STR56## wherein:X is a substituted or unsubstituted C₁ -C₆ alkyl group; andB is glutamicacid or a derivative thereof, linked through the amino portion to forman amide.
 8. A compound according to claim 7, wherein when X issubstituted, the substituents are selected from OH, NH₂, O-methyl,O-ethyl, SH, SCH₃ and NH-methyl.
 9. A compound according to claim 7,wherein X is unsubstituted.
 10. A compound according to claim 9, whereinX is methyl or ethyl.
 11. A compound according to claim 7, wherein B isdiethyl glutamate.